Sistem Transfer of HPLC Methods to UHPLC

8/11/2019 Sistem Transfer of HPLC Methods to UHPLC

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Higher Efficiencies Mean Increased Sample ThroughputUltra High Pressure Liquid Chromatography (UHPLC) is based on a modification to traditional liquidchromatographic systems that makes possible the use of increased operating pressures. Higher maxi-mum operating pressures mean that packing materials consisting of small (


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Equation 1 Adjusted column length can

easily be calculated when scaling from

HPLC to UHPLC.

Equation 2 Changing column dimensions

requires an adjusted injection volume.

Equation 3 Changing column internal

diameter requires using an adjusted

flow rate.

Equation 4 When scaling down a gradient

method, the time program needs to be

adjusted.

Adjusting the Column DimensionsWhen performing a scale-down procedure, a few simple calculations can beused to determine equivalent run conditions. The first calculation to make isthe determination of the appropriate column length. Keeping the same col-umn length while decreasing the particle size will increase the number oftheoretical plates in that given column length. Therefore, when decreasingparticle size, column length can be shortened without losing resolution. Byadjusting the column length properly, using Equation 1,we can maintain the

same separation.

Adjusting the Injection VolumeOnce we have determined the proper column length, we can determine theappropriate injection volume. Decreasing the column internal diameter andlength, decreases the overall column volume and sample capacity. Therefore,we must alter the injection volume as described in Equation 2. Please notethat since overall column volume has decreased, it is important to match thesample solvent to the starting mobile phase composition. Mismatched sam-ple solvents can cause irreproducible retention times, efficiencies, and evenchanges in selectivity. If using a larger injection volume than calculated,check for peak abnormalities and irreproducibility that could result fromphase overload.

Adjusting the Flow Rate

Next, when decreasing the internal diameter of the columns from 4.6mm to2.1mm, the new column flow rate needs to be determined. Linear velocity isdefined as the distance mobile phase travels over time (cm/min.), whereasflow rate is the volume of mobile phase that travels over time (mL/min.). Tomaintain the same linear velocity through a column with a smaller internaldiameter, the flow rate must be decreased proportionally to the columninternal diameter. Equation 3 can be used to determine the adjusted flow rateneeded to maintain equivalent mobile phase linear velocities when changingcolumn internal diameter.

Thus far, our example has focused on establishing a UHPLC method withequivalent linear velocity. However, a significant advantage of using a


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Figure 2 Converting the conventional HPLC method to UHPLC, using the calculations given in Equations 1-4,

reduces analysis time.

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A. Pinnacle DB Biphenyl1.9m, 50 x 2.1mm

B. Pinnacle DB Biphenyl5m, 150 x 4.6mm

Scaling downmethods savesanalysis time!

Sample:Inj.: 10LConc.: 100g/mLSample diluent: starting mobile phase (80:20 A:B)

Column: Pinnacle DB BiphenylCat.#: 9409565Dimensions: 150mm x 4.6mmParticle size: 5mPore size: 140

Conditions:Mobile phase: A: 0.1% formic acid in water

B: 0.1% formic acid in acetonitrile

Time(min.) %B0.0 201.0 206.0 808.0 80

Flow: 1.0mL/min.Temp.: 30CDet.: UV @ 254nm

LC_PH0462

LC_PH0461

ConclusionAfter determining the equivalent conditions for scaling down the analysis of sulfonamides, we can see that the separations areequivalent, while the analysis time was greatly reduced (Figure 2). Under conventional HPLC the last compound eluted at 7.2minutes and under UHPLC the last compound eluted at 2.6 minutes. By following the procedure described here to ensure thatthe columns are equivalent, scaling analytical procedures from HPLC to UHPLC, or vice versa, can easily be accomplished.

LC_PH0460

C18 selectivityunder sameconditions

More selectivity for earlyeluting compounds.

Sample:Inj.: 1LConc.: 100g/mlSample diluent: starting mobile phase (80:20 A:B)

Column: 1.9m Pinnacle DB BiphenylCat. #: 9409252Dimens ions: 50 x 2 .1mmPart ic le s ize: 1 .9mPore size: 140

Conditions:Mobile phase: A: 0.1% formic acid in water

B: 0.1% formic acid in acetonitrileTime(min.) %B0.0 20

0.4 202.1 802.8 80

Flow: 0.2mL/min.Temp.: 30CDet.: UV @ 254nm

Peak List:1. sulfadiazine2. sulfathiazole3. sulfamerazine4. sulfamethazine5. sulfachlorpyridizine6. sulfamethoxazole7. sulfamethoxine

Peak List:1. sulfadiazine2. sulfathiazole3. sulfamerazine4. sulfamethazine5. sulfachlorpyridizine6. sulfamethoxazole7. sulfamethoxine

Time (min)


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Restek Trademarks:Pinnacle, Restek logo.

1.9m Pinnacle DB HPLC Columns

Chromatographic Properties:Highly base-deactivated spherical silica manufactured by Restek.

particle size: 1.9mpore size: 140endcap: yes

pH range: 2.5 to 7.5temperature limit: 80C

Physical Characteristics:

Ruggedness and reproducibility are guaranteed, as we control every step in the process, from basesilica to bonded phase to final packed column. The silica particles are classified and selected to give anexceptionally tight distribution around 1.9m, while eliminating

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Sistem Transfer of HPLC Methods to UHPLC